A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2

Suarez, Stéphanie; Pieren, Michel; Cariolato, Luca; Arn, S.; Hoffmann, Ute; Bogucki, Augustyn; Manlius, C.; Wood, Jeanette M.; Ballmer-Hofer, Kurt

doi:10.1007/s00018-006-6254-9

Cited by 169 publications

(201 citation statements)

References 64 publications

(65 reference statements)

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“…NRP1 binding to VEGF-A is isolated to C-terminal portion of VEGF-A165a and VEGF-A165a exon 8a encoded residues are indispensible for NRP1 binding. 9 , 18 , 23 , 24 , 60 Crystallographic studies are not yet available for intact VEGF-A121a, but this ligand has the entire exon 5 and exon 8a encoded sequence intact and is shown by the SPR technique (cell-free) to bind NRP1, with lower affinity compared to VEGF-A165a 18 , 27 and to NRP2 with similar affinity as NRP1. 18 Modulation of VEGF-A121a-VEGFR2 signaling by the expression of (or interference with) NRP1 on cells in vitro suggests that these components are interacting (or at least interdependent), although it is also established that VEGF-A121a cannot form the extracellular VEGFR2-VEGF-NRP1 bridge that VEGF-A165a is capable of forming (or if a weak extracellular VEGFR2-VEGF-A121a-NRP1 bridge exists, it can not be measured experimentally).…”

Section: Discussionmentioning

confidence: 99%

“…VEGF-A159), or have their exon 8a replaced by exon 8b (8a: CDKPRR; 8b: SLTRKD; i.e. VEGF-A121b and VEGF-A165b), do not bind NRP1 at all (figure 4 of 23 ). They conclude that exon 8a-encoded sequence is required for NRP1 binding, that the heparin-binding domains (exons 6 and/or 7) are not sufficient for NRP1 binding, and that exon 8a is necessary for proper folding of the heparin-binding domains, as VEGF-A165b has reduced HS binding, no NRP1 binding and altered (delayed and attenuated) signaling characteristics via VEGFR2 and ERK kinases.…”

Section: Evidence That the Exon 8a Domain Which Vegf-a121a Has Is Rmentioning

confidence: 99%

“…They conclude that exon 8a-encoded sequence is required for NRP1 binding, that the heparin-binding domains (exons 6 and/or 7) are not sufficient for NRP1 binding, and that exon 8a is necessary for proper folding of the heparin-binding domains, as VEGF-A165b has reduced HS binding, no NRP1 binding and altered (delayed and attenuated) signaling characteristics via VEGFR2 and ERK kinases. 9 , 10 , 23 Only VEGF-A isoforms containing the exon 8a-encoded sequence (VEGF-A xxx a; Fig. 1) compete with VEGF-A165a for binding to NRP1.…”

Section: Evidence That the Exon 8a Domain Which Vegf-a121a Has Is Rmentioning

confidence: 99%

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VEGF-A121a binding to Neuropilins – A concept revisited

Sarabipour

Gabhann

2017

Cell Adhesion & Migration

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All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue1, and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs.2 There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms – that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a – bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not ‘bridge’ VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane (TM) and intracellular (IC) domains.

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Section: Discussionmentioning

confidence: 99%

Section: Evidence That the Exon 8a Domain Which Vegf-a121a Has Is Rmentioning

confidence: 99%

Section: Evidence That the Exon 8a Domain Which Vegf-a121a Has Is Rmentioning

confidence: 99%

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VEGF-A121a binding to Neuropilins – A concept revisited

Sarabipour

Gabhann

2017

Cell Adhesion & Migration

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“…An alternate splice form of VEGF, VEGF165b, differs only in the sequence of the final six residues owing to switching of exon 8 with exon 9. VEGF165b contains all of exon 7, but does not interact with Nrp and shows dramatically reduced angiogenic potential (43). In particular, VEGF165b has a C-terminal Asp in contrast to the critical C-terminal Arg of VEGF165 (44) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for ligand and heparin binding to neuropilin B domains

Kooi

Jusino

Perman

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

206

252

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Neuropilin (Nrp) is a cell surface receptor with essential roles in angiogenesis and axon guidance. Interactions between Nrp and the positively charged C termini of its ligands, VEGF and semaphorin, are mediated by Nrp domains b1 and b2, which share homology to coagulation factor domains. We report here the crystal structure of the tandem b1 and b2 domains of Nrp-1 (N1b1b2) and show that they form a single structural unit. Cocrystallization of N1b1b2 with Tuftsin, a peptide mimic of the VEGF C terminus, reveals the site of interaction with the basic tail of VEGF on the b1 domain. We also show that heparin promotes N1b1b2 dimerization and map the heparin binding site on N1b1b2. These results provide a detailed picture of interactions at the core of the Nrp signaling complex and establish a molecular basis for the synergistic effects of heparin on Nrp-mediated signaling.semaphorin ͉ Tuftsin ͉ VEGF N europilins (Nrps) are essential cell surface receptors with central roles in both angiogenesis and axon guidance (1-3). During angiogenesis, Nrp directly binds VEGF and functions as a coreceptor for VEGF along with VEGF receptor (VEGFR)-2, one of the three VEGFR tyrosine kinases (3, 4). During neural development, Nrp directly binds semaphorin and functions as a semaphorin coreceptor with members of the plexin family (5). Additionally, interactions with both neural adhesion protein L1 and Nrp-interacting protein (NIP), have been shown to be involved in a variety of other cellular processes (6-8).Nrp plays a stimulatory role in angiogenesis, a process critical for growth of solid tumors (reviewed in refs. 4 and 9-11). Nrp expression is observed in tumor vasculature, and overexpression promotes tumorigenesis in vivo for a variety of solid tumors including pituitary, prostate, breast, and colon cancers (12-15). In contrast, a soluble splice form containing only part of the extracellular domain of Nrp inhibits tumorigenesis (16) as do a number of peptides that block VEGF binding to Nrp (17,18). Recent evidence has also demonstrated a role for Nrp in hematological malignancies. Nrp overexpression is observed in both multiple myeloma and acute myeloid leukemia and, in the latter case, is associated with significantly reduced survival (19,20). Strategies to inhibit Nrp activity are thus being developed as potential antitumor therapies (reviewed in ref. 11).Higher eukaryotes possess two Nrp homologs, Nrp-1 and Nrp-2, which share 44% amino acid sequence identity (1). Nrp extracellular regions are composed of two complement binding CUB domains (a1 and a2) followed by two coagulation factor domains (b1 and b2), a MAM (meprin, A5, ) domain (c1), a single membrane-spanning region, and a short cytoplasmic tail (Fig. 1A) (21, 22). The a1 and a2 domains of Nrp are essential for binding to the core seven-bladed Sema domain of semaphorin as well as contributing to interactions with VEGF (23, 24). The coagulation factor domains b1 and b2 contain the high-affinity binding site for the basic heparin binding domain (HBD) of VEGF165 as well a...

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“…In addition, VEGF-C promotes the formation of VEGFR-2/VEGFR-3 heterodimers whose signaling potential is not yet clear (9). VEGF signaling is modulated through interactions with distinct heparan sulfate proteoglycans and neuropilins, which act as coreceptors (10)(11)(12)(13)(14). VEGFs exist in multiple isoforms that are generated by alternative splicing and posttranslational processing and display distinct receptor specificities (15).…”

mentioning

confidence: 99%

Structural determinants of growth factor binding and specificity by VEGF receptor 2

Leppänen

Prota

Jeltsch

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

159

207

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Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 2∶2 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

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A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2

Abstract: Abstract. The development of functional blood and lymphatic vessels requires spatio-temporal coordination of the production and release of growth factors such as vas-

Cited by 169 publications

References 64 publications

VEGF-A121a binding to Neuropilins – A concept revisited

VEGF-A121a binding to Neuropilins – A concept revisited

Structural basis for ligand and heparin binding to neuropilin B domains

Structural determinants of growth factor binding and specificity by VEGF receptor 2

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